The most serious form of bacterial food poisoning is botulism which is caused by neurotoxins produced by Clostridium botulinum. The toxins are usually preformed by the causative organism in foods and subsequently absorbed through the intestinal tract and transported via the circulatory system to motor nerve synapses where their action blocks normal neural transmissions. Various serotypes of C. botulinum produce neurotoxins with similar toxic activity but which differ antigenically. Serotype A toxin is the predominant cause of botulism in the United States while type B toxin is the most prevalent in Europe.
Crystalline type A botulinum toxin complex was prepared in 1979 by E. J. Schantz of the Food Research Institute/Department of Food Microbiology and Toxicology at the University of Wisconsin-Madison. It has been used medically to treat hyperactive muscle disorders such as strabismus, blepharospasm, and spasmodic torticollis. Treatment involves injection of nanogram quantities of the toxin directly into the hyperactive muscles. The toxin inhibits the release of acetylcholine across the synaptic junction causing a decrease in the activity of the injected muscles.
Type A neurotoxin produced by C. botulinum is present as part of a complex of at least seven different noncovalently bound proteins. High quality type A toxin complex has a specific toxicity of 3.times.10.sup.7 mouse intraperitoneal 50% lethal doses (LD.sub.50) per mg. The purified neurotoxin, that is the neurotoxin that has been chromatographically separated from the other proteins of the toxin complex, has a specific toxicity of 9.times.10.sup.7 to 1.times.10.sup.8 LD.sub.50 per mg. In the medical field, a unit (U) is considered to be 1 LD.sub.50. Toxin titers are determined in female, white mice, 18-22g in weight according to the method of Schantz and Kautter as described in Association of Official and Analytical Chemistry, vol. 61, p. 96, (1978).
A major drawback to the use of botulinum toxin in treatment of hyperactive muscle disorders is development of antibodies or other types of immunity by patients. The toxin is recognized by patient's immune systems as foreign and stimulates antibody production. This renders treatment of the various hyperactive muscle disorders with botulinum toxin ineffective. One way to reduce the number of patients developing neutralizing antibodies would be to develop a more shelf-stable product with a higher specific activity following lyophilization. Such a formulation would result in a product that is not as antigenic as the currently available product and lesser quantities of toxin would be required for treatment.
Botulinal toxin is very susceptible to denaturation due to surface denaturation, heat, and alkaline conditions. Lyophilization or freeze-drying of botulinal toxin is the most economically sound and practical method of distributing the product in a form that is stable and readily used by the clinician. The current commercial type A botulinal toxin product is made by combining up to 500 ng/ml of type A toxin complex in 5.0 mg/ml human serum albumin (HSA) with 9.0 mg/ml sodium chloride at a pH of 7.3. After dissolution, 0.1 ml is dried to obtain 100.+-.30 active U of toxin, 0.5 mg of HSA, and 0.9 mg of sodium chloride per vial. This product has a saline concentration of 0.9% when reconstituted in 1.0 ml of dH.sub.2 O. The current commercial formulation which employs the toxin complex has a specific toxicity of about 2.5 U/ng after drying BOTOX.RTM., crystalline botulinum toxin complex, Allergon, Inc. of Irvine, Calif. The considerable loss (up to 90%) of activity during drying causes the formation of inactive toxin that serves as a toxoid inciting antibody formation.
A rabbit model in which repetitive injections of various type A toxin preparations have been given to simulate the treatment of a focal dystonia has been used to assess the immunogenicity of various toxin preparations. The model consists of injecting albino rabbits with sublethal doses of the toxin over a period of time and assaying the serum of the animals for the ability to neutralize a small but carefully quantitated amount of purified type A toxin. Our results show that the product presently available in the United States which has the lowest specific toxicity of all preparations tested is the most antigenic of all the preparations tested to date. These results indicate that high specific activity preparations reduce the probability of patients developing neutralizing antibodies. It obviously would be desirable to have higher specific activity preparations than those currently available.
The current commercial product must be stored at a temperature of -10.degree. C. or less to maintain the labelled potency for the one year shelf life. It also would be advantageous to have a product that could be stored at higher temperatures (i.e. room temperature). This would facilitate more practical shipping and storage of the toxin.